Methods for Segregating Particles Using an Apparatus with a Size-Discriminating Separation Element Having an Elongate Leading Edge

ABSTRACT

The disclosure relates to an apparatus for segregating particles on the basis of their ability to flow through a stepped passageway. At least some of the particles are unable to pass through a narrower passageway bounded by a segregating step, resulting in segregation of the particles. The breadth of the leading edge of at least one step of the apparatus is significantly greater than the overall width of the passageway in which the step occurs, permitting high and rapid sample throughput. The apparatus and methods described herein can be used to segregate particles of a wide variety of types. By way of example, they can be used to segregate circulating tumor cells from a human blood sample.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/077,811, filed 12 Nov. 2013, which claims priority to U.S.provisional patent application 61/794,468, filed 15 Mar. 2013. Theforegoing applications are incorporated herein by reference.

BACKGROUND OF THE DISCLOSURE

Mechanical devices intended for manipulation of biological cells andother small particles and having structural elements with dimensionsranging from tens of micrometers (the dimensions of biological cells) tonanometers (the dimensions of some biological macromolecules) have beendescribed. For example, U.S. Pat. No. 5,928,880, U.S. Pat. No.5,866,345, U.S. Pat. No. 5,744,366, U.S. Pat. No. 5,486,335, and U.S.Pat. No. 5,427,946 describe devices for handling cells and biologicalmolecules. PCT Application Publication No. WO 03/008931 describes amicrostructure for particle and cell separation, identification,sorting, and manipulation.

U.S. Pat. No. 7,993,908 describes a microscale apparatus for separatingcells and other particles based on their size. The apparatus describedin that patent includes a stepped separation element interposed betweentwo regions of a void formed by a cover and body, and separation ofparticles within the apparatus is governed by the ability of particlesinitially present in one region to traverse the stepped separationelement to arrive at the other region. The subject matter disclosedherein is considered an improvement upon this apparatus.

The subject matter disclosed herein can be used to segregate andmanipulate biological cells, organelles, cell conglomerates, and otherparticles from mixed populations of particles or cells.

SUMMARY OF THE DISCLOSURE

The present disclosure relates to an apparatus for segregating smallerand larger particles. The apparatus includes a body and a cover thatdefine a void between them. The void contains a separation element thatsegregates an inlet region and an outlet region of the void. Togetherwith one or more surfaces of the void, the separation element defines achannel that fluidly connects the inlet and outlet regions by way of aseparating portion. The channel has an overall width at the separatingportion and a height defined by the distance between the separationelement and the surface of the void. At least one of the body, thecover, and the separation element bears a segregating step disposedwithin and having a leading edge extending substantially completelyacross the separating portion of the channel. The channel is dividedinto an upstream portion on the inlet side of the leading edge and asubstantially lamellar downstream portion on the outlet side of theleading edge. The height of the upstream portion is sufficient tofacilitate passage therethrough of both larger and smaller particles.The height of the downstream portion is sufficient large to facilitatepassage therethrough of the smaller particles and sufficiently small toinhibit passage therethrough of the larger particles. The breadth of theleading edge is substantially greater than the overall width of thechannel at the separation region (which is normally the same width asthat of the segregating step, meaning that leading edge of thesegregating step is normally longer than the width of that step). Theparticles can be segregated by passing them through the channel andrecovering particles based on their ability to traverse the segregatingstep.

In one embodiment, the upstream portion of the channel is substantiallylamellar, meaning that it is defined by two broad surfaces that aresubstantially parallel to one another.

The breadth of the leading edge can be substantially (e.g., at least 100times) greater than a characteristic dimension of the larger particles,so that many such particles can be trapped at the leading edge withoutsubstantially preventing bulk fluid flow past the leading edge. Thebreadth of the leading edge can also be substantially (e.g., at least1.5, 2, 3, 4, 5, 10, 20, 50, 100, 500, 1000, 10000, or 100000 times)greater than the overall width of the channel at the separation region(or the width of the segregating step within the channel). By way ofexample, the height of the downstream portion (i.e., the portion of thechannel of the leading edge of the segregating step) can be selected sothat it is sufficiently small to inhibit passage therethrough of aselected cell type (e.g., a circulating tumor cell or human fetalstem-like cells), sufficiently large that it does not inhibit passagetherethrough of a selected cell type (e.g., human red blood cells), or acombination of these.

The leading edge of any segregating step can have an angular, curved,undulating, invaginated, or irregular shape. The segregating step canhave, on its inlet side, an upstream face that is substantiallyperpendicular to the portion of the step that defines the downstreamportion of the channel.

The separation element can be integral with at least one of the body andthe cover. It can also be a separate item interposed between the bodyand the cover. The device can have one or more supports for maintainingthe height of the channel. Such supports can be disposed within thechannel and extend between the separation element and the surface of thevoid, for example.

The disclosure also relates to methods of segregating larger and smallerparticles. These methods include providing a fluid suspension of largerand smaller particles at the inlet of the apparatus described herein.Fluid is urged through the channel and one can collect at least one ofi) smaller particles (e.g., red blood cells) at the outlet region, andii) larger particles (e.g., circulating tumor cells) upstream of theleading edge of the segregating step.

The disclosure also relates to methods of diagnosing occurrence of atumor in a vertebrate subject. These methods include steps of i)providing a blood sample obtained from the subject to the inlet regionof the apparatus described herein (the height of the lamellar portion ofthe downstream portion of the channel is smaller than the size of aCTC), passing the sample through the channel of the apparatus, andthereafter examining the portion of the apparatus upstream of theleading edge of the segregating step for the presence of a cell.Presence of at least one cell is an indication that a tumor occurs inthe subject. One or more diagnostic tests can thereafter be used toassess a characteristic of a tumor cell for at least one cell that waspresent upstream of the leading edge of the segregating step afterpassing the sample through the channel. Examples of such tests includebinding the cell or an extract thereof with a tissue-specific ortumor-specific antibody, analyzing nucleic acids obtained from such acell that was present upstream of the leading edge, or assessing theproliferative capacity of the cell.

The disclosure further relates to methods of assessing the efficacy of atumor treatment for a subject afflicted with a tumor. These methodsinclude isolating CTCs from blood samples obtained from the subjectbefore and after the treatment using the methods described herein. Atleast one characteristic of the CTCs isolated from the samples iscompared among the samples. A difference in the characteristics of CTCs(e.g., CTC concentration or number) isolated from the blood samples isan indication of the efficacy of the treatment.

The disclosure also relates to methods of reducing CTC load in avertebrate subject. Such methods include steps of i) providing bloodobtained from the subject at the inlet of the apparatus described herein(wherein the height of the lamellar portion of the downstream portion ofthe channel is smaller than the size of a CTC), ii) urging the bloodthrough the channel to deplete CTCs from the blood, iii) collectingCTC-depleted blood at the outlet region, and iv) returning theCTC-depleted blood to the subject.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings.

These drawings are included for the purpose of illustrating thedisclosure. The disclosure is not limited to the precise arrangementsand instrumentalities shown.

FIG. 1 consists of FIGS. 1A, 1B, 1C, and 1D, and illustrates a prior artseparation element 1 having two integral, rectangular slab-shaped steps,including a focusing step 10 and a segregating step 11. Devices having aseparation element of this sort are disclosed in U.S. Pat. No.7,993,908, for example. FIG. 1A is an elevated view of the separationelement 1 in which the rectangular shape of the face 20 of the focusingstep 10 can be seen adjacent the broad face 40 thereof and therectangular shape of the face 21 of the segregating step 11 can be seenadjacent the broad face 41 thereof. FIG. 1B is a side view of theseparation element 1 shown in FIG. 1A, showing the height differencebetween the focusing and segregating steps (10 and 11, respectively).FIG. 1C is an orthogonal view of the separation element 1 shown in FIGS.1A and 1B. FIG. 1D is a cross-sectional view of the separation element 1disposed in a fluid channel defined by a gap between a cover 4 and thebody 2 of an apparatus described herein. In FIG. 1D, the height (h₁) ofa downstream portion of the fluid channel, the height (h₀) of anupstream portion of the fluid channel, and the height (h_(c)) of thefluid channel itself are shown. The height (h₁) of the downstreamportion is defined by the distance between the segregating step 11 andthe cover 4, and the height (h₀) of the upstream portion is defined bythe distance between the focusing step 10 and the cover 4.

FIG. 2 consists of FIGS. 2A, 2B, and 2C and illustrates a separationelement 1 having a rectangular slab-shaped focusing step 10 and asegregating step 11 having a slab shape but having an undulating face 21and leading edge 31. FIG. 2A is an elevated view of the separationelement 1 in which the undulating shape of the face 21 of thesegregating step 11 can be seen adjacent the broad face 41 thereof. FIG.2B is a side view of the separation element 1 shown in FIG. 2A, showingthe height difference between the focusing and segregating steps (10 and11, respectively). FIG. 2C is an orthogonal view of the separationelement 1 shown in FIGS. 2A and 2B.

FIG. 3 consists of FIGS. 3A, 3B, and 3C and illustrates a separationelement 1 having a rectangular slab-shaped focusing step 10 and asegregating step 11 having a slab shape but having an irregular face andleading edge. FIG. 3A is an elevated view of the separation element 1 inwhich the rectangular shape of the face of the focusing step 10 can beseen adjacent the broad face 40 thereof and the irregular shape of theface of the segregating step 11 can be seen adjacent the broad face 41thereof. FIG. 3B is a side view of the separation element 1 shown inFIG. 3A, showing the height difference between the focusing andsegregating steps (10 and 11, respectively). FIG. 3C is an orthogonalview of the separation element 1 shown in FIGS. 3A and 3B.

FIG. 4 consists of FIGS. 4A, 4B, and 4C, and illustrates a separationelement 1 having a rectangular focusing step 10 and three steps atop itand downstream (relative to BFF) from its leading edge. Each of thefirst segregating step 11, second segregating step 12, and thirdsegregating step 13 has a chevron-shaped leading edge (leading edges 31,32, and 33, respectively. Bulk fluid flow BFF direction is indicated.FIG. 4A is an elevated view of the separation element 1. FIG. 4B is aside view of the separation element 1 shown in FIG. 4A, showing theheight differences among the steps. A recessed portion of the separationelement 1 downstream of steps 11-13 forms part of an outlet passagewayby way of which material that has traversed all of steps 10-13 can becarried away from the separation element 1. FIG. 4D is a cross-sectionalview of the separation element 1 disposed in a fluid channel defined bya gap between a cover 4 and the body 2 of an apparatus described herein.In FIG. 4D, the heights (h₃, h₂, and h₁, respectively) of serialdownstream portions of the fluid channel, the height (h₀) of an upstreamportion of the fluid channel, and the height (h_(c)) of the fluidchannel itself are shown. The height (h₃) of a third downstream portionis defined by the distance between the third segregating step 13 and thecover 4. The height (h₂) of a second downstream portion is defined bythe distance between the second segregating step 12 and the cover 4. Theheight (h₁) of a first downstream portion is defined by the distancebetween the first segregating step 11 and the cover 4. The height (h₀)of the upstream portion is defined by the distance between the focusingstep 10 and the cover 4.

FIG. 5 consists of FIGS. 5A and 5B and illustrates a separation element1 having a focusing step 10 having a curved transitional face 20 thatextends completely across the separation element 1 and three segregatingsteps atop it and downstream (relative to BFF) from the focusing step10. Each of the first segregating step 11, second segregating step 12,and third segregating step 13 has a curved leading edge, meaning thatthe breadth of the leading edge of each of segregating steps 11-13 isgreater than its width (unlike the length of the leading edge 30 offocusing step 10, which is equal to its width). Bulk fluid flow BFFdirection is indicated. FIG. 5A is an elevated view of the separationelement 1. FIG. 5B is a side view of the separation element 1 shown inFIG. 5A, showing the height differences among the steps. A recessedportion of the separation element 1 downstream of steps 10-13 forms partof an outlet passageway by way of which material that has traversed allof steps 10-13 can be carried away from the separation element 1.

FIG. 6 consists of FIGS. 6A and 6B and illustrates a separation element1 having a rectangular focusing step 10 and three segregating steps atopit and downstream (relative to BFF) from its leading edge. Each of thefirst segregating step 11, second segregating step 12, and thirdsegregating step 13 has a serpentine leading edge. Bulk fluid flow BFFdirection is indicated. FIG. 6A is an elevated view of the separationelement 1. FIG. 6B is a side view of the separation element 1 shown inFIG. 6A, showing the height differences among the steps.

FIG. 7 consists of FIGS. 7A, 7B, 7C, and 7D (which are drawnapproximately to scale relative to one another) and illustrates fourstep configurations having equal breadth (B) in a fluid channelindicated by heavy lines. The direction of bulk fluid flow (BFF) isindicated, and step height increases from the upstream to the downstreamside of the step, which is indicated by a line extending across thefluid channel in the figures. In FIG. 7A, step height rises across halfthe fluid channel at a relatively upstream position and across the otherhalf of the fluid channel at a relatively downstream position, with thestep face extending between those two positions. The length (L) of theextended step face is equal to four times the width (W) of the fluidchannel in FIG. 7A, yielding a total B of the step equal to 5W. In FIG.7B, the step has two portions extending between an upstream position anda downstream position. Although the length of the step face extension inthe direction of BFF is only 2W, there are two such extensions. As aresult the total breadth of the face in FIG. 7B is 2×2W+W, or 5W.Similarly, the step shown in FIG. 7C, which has three portions extendingbetween upstream and downstream positions (i.e., four step faceextensions of length W) exhibits a B of 4×W+W, or 5W. The step shown inFIG. 7D, which has five portions extending between upstream anddownstream positions (i.e., eight step face extensions of length W/2)exhibits a B of 8×W/2+W, or 5W. Of note, L of steps having equal Bdecreases with increasing invagination of the steps. This illustratesthat miniaturization of particle separation functionality of a step canbe achieved by increasing the complexity (B/L) of the step face.

FIG. 8 is an embodiment of a particle segregation apparatus as describedherein constructed to have a size approximately equal to a commonmicroscope slide. Inlet and outlet regions 52 and 58 are shown, as isthe separation portion 55 of the channel that extends between inlet andoutlet regions 52 and 58.

FIG. 9 is a magnified image of PC3 prostate cancer cells captured usinga segregation apparatus described herein. In the image, cells can beseen on or upstream (bulk fluid flow is from left to right in theFigure) from the first segregation step 11 and the second segregationstep 12, while few or no cells are present on focusing steps 10.

FIG. 10 consists of FIGS. 10A and 10B. Each of these is a magnifiedimage of PC3 prostate cancer cells captured using a segregationapparatus described herein. In each image, cells can be seen on orupstream (bulk fluid flow is from left to right in the Figure) from thefirst segregation step 11, the second segregation step 12, and the thirdsegregation step 13, while few or no cells are present on focusing steps10.

DETAILED DESCRIPTION

The disclosure relates to an apparatus for segregating particles on thebasis of their ability to traverse a passageway. Particles (e.g.,particles suspended in a liquid or gaseous fluid or particles in avacuum) are moved through a stepped passageway 55 defined by aseparation element 1 in the apparatus. The stepped passageway 55connects portions of a void 50 defined by a body 2 and a cover 4, andthe separation element 1 is present within the void 50 and separatesinlet and outlet regions (52 and 58, respectively) regions of the void50. The separation element 1 may be a discrete element, or it may beattached to or integral with one of body 2 and cover 4.

The stepped passageway 55 fluidly connects the inlet region 52 and theoutlet region 58 of the void 50, and contains at least one segregatingpassageway 101 that has a narrow dimension defined by the distancebetween the face 41 of a (first) segregating step 11 and another portionof the (first) segregating passageway 101, such as the face of the body2 or the cover 4. Only some particles in the fluid are able to move intothe segregating passageway 101. The net result is that some particlescan move through the entire stepped passageway 55, while other particlesare retained within the apparatus, such as upstream of the segregatingpassageway 101. Segregation of particles is thus achieved. Movement ofparticles can be motivated by fluid flow, gravity, vibration, or anycombination of these, for example.

In contrast to analogous devices disclosed, for example, in U.S. Pat.No. 7,993,908 (illustrated in FIG. 1), the leading edge 31 andtransitional face 41 of at least one of the segregating steps of thedevices described herein has a breadth substantially greater (e.g., by afactor of at least 1.5, 2, 10, 25, 100, 1000, 10,000, or 100,000) thanthe width of the segregating step (i.e., greater than the width of thestepped passageway 55 in which the segregating step 11 is disposed.Because separation of particles in bulk fluid flowing past a segregatingstep 11 tends to occur mostly at the leading edge and face 21 of thestep, increasing the breadth of these, relative to the width of thesegregating step 11 and passageway can have several beneficial effects.

Particles flowing past a segregating step 11 in a bulk fluid willnecessarily have a size, in at least one dimension, not greater than theheight of the segregating passageway 101 above that segregating step 11(i.e., the narrow dimension of the segregating passageway; otherwise theparticles would be unable to pass therethrough with the bulk fluid).Likewise, particles having dimensions greater than the height of thesegregating passageway 101 above a segregating step 11 will cease toflow with bulk fluid at or near the leading edge 31 or the transitionalface 21 of the segregating step 11 and will tend to accumulate there.Increasing the breadth of the transitional face 21 and leading edge 31beyond the overall width of the passageway in which the segregating step11 is disposed permits multiple particles to be accommodated at theleading edge 31 or elsewhere along the transitional face 21 (e.g., ifthe face is sloped). Thus, the apparatus in which the leading edge has abreadth greater than the overall width of the segregating passageway 101can be used to capture one or more size-segregated particles at or nearthe leading edge 31 of the segregating step 11. As the breadth of theleading edge 31 of the segregating step 11 increases, a greater numberof size-segregated particles can be captured at the transitional face 21thereof without clogging the device. It is desirable that the ratio ofthe breadth of a segregating step be substantially greater (e.g., atleast 1.5-fold, and more preferably 2-, 3-, 4-, 5-, 10-, 20-, 50-, 100-,500-, or 1000-fold greater) than the width of the passageway that boundsthe ends of the leading edge of the segregating step.

In order to accommodate a leading edge 21 having a breadth greater thanthe width of the segregating passageway 101, the leading edge 21 and thetransitional face 31 of a segregating step 11 must not extend straightacross the narrowest width of the segregating passageway 101. Theleading edge can be straight (e.g., extending obliquely across thepassageway 101 in a direction other than the narrowest dimensionthereof) or composed of multiple straight segments (see FIGS. 4 and 7).The leading edge 21 can also be curved (See FIG. 5), invaginated (SeeFIGS. 2, 3, and 6), or meandering (See FIG. 3) in shape, therebyincreasing its breadth (and that of its corresponding segregating step11 and transitional face 21) relative to the width of the segregatingpassageway 101. As a result of such leading edge shapes, the capturecapacity of the apparatus can be increased, relative to prior artdevices in which segregating steps 11 extended directly across the widthof the segregating passageway 101.

In one embodiment, the leading edge of the step is highly curved (e.g.,has many invaginations, such as the invagination shown in segregatingsteps 11-13 in FIG. 6), so that its breadth is significantly greaterthan the overall width of the passageway in which the step is contained.By way of example, FIGS. 4, 5, and 6 illustrate a four-step separationelement 1 that can be accommodated within a passageway having asubstantially rectangular cross section. In FIGS. 4, 5, and 6, theseparation element 1 has an overall width equal to the width (i.e., inthe direction perpendicular to bulk fluid flow BFF) of the segregatingpassageway 101. The separation element 1 in each of these figuresincludes a focusing step 10 that extends directly across the segregatingpassageway 101 (like steps in prior art devices) and thus has a breadthequal to the overall width of the passageway.

In FIG. 4, the leading edge of each of segregating steps 11-13 has abreadth greater than the overall width of the segregating passageway101—if the vertex of the chevron-shaped leading edge of each step is aright angle, then the length of the leading edge of each step is (byapplication of the Pythagorean equation) equal to twice the square rootof (the square of the width of the passageway divided by two) (i.e., ifthe width of the segregating passageway 101 is one unit, then thebreadth of each step is about 1.4 units).

In FIG. 5, the breadth of the leading edge of each of segregating steps11-13 is greater than the overall width of the segregating passageway101, on account of the curvature of the leading edge of each step.

In FIG. 6, the breadth of the leading edge of each of segregating steps11-13 is longer still, owing to the curvature and invagination of eachstep.

The geometries shown in FIGS. 4-6 are for illustrative purposes. Stepleading edges can have innumerable geometric shapes. The shapes shown inthose figures simply illustrate the concept that increasing thecomplexity (especially ‘folding’ or invagination) of the leading edgecan cause the breadth of the leading edge of any step to greatly exceedthe overall width of the passageway within which the step occurs. Inanother embodiment of the separation element shown in FIGS. 4-6, theseparation element lacks focusing step 10 and the segregating steps11-13 are integral with three adjacent walls of the substantiallyrectangular segregating passageways 101-103 in which the separationelement 1 is disposed.

Particles unable to traverse a segregating step can be urged in thedirection of bulk fluid flow along the leading edge of the segregatingstep. Thus, for example, particles that are able to traverse thefocusing step, but are unable to traverse the segregating steps of thedevice shown in FIG. 6 will tend to be urged by bulk fluid flow towardthe central invagination in the segregating steps and toward theperipheral edges of those steps. Although not shown, the shapes of theleading edges of the segregating steps illustrated in FIGS. 4 and 5 canbe inverted relative to the direction of BFF shown in those figures(i.e., so that the apices of the chevron-shaped and curved steps liedownstream from the edges of the steps). Steps can thus be shaped tofacilitate or promote accumulation of particles at selected locationsalong their leading edges.

Particles captured at the leading edge 31 or along the transitional face21 of a segregating step 11 (i.e., a step past which some, but not all,particles in a bulk fluid can move with the bulk fluid flowing past thestep) will tend to occlude fluid flow past the step at or on which theyare captured (i.e., at the position at which their movement with thebulk fluid stops or is substantially inhibited). If captured particlesocclude fluid flow past a substantial portion(e.g., >0.01%, >0.1%, >1%, >10%, >50%, >90%, or >99%) of the steppedpassageway, this will decrease the throughput of the segregatingpassageway 101 (i.e., the volume of fluid that can be passed through thenarrow passageway in a unit of time at a selected fluid pressure dropacross the step) can be significantly diminished.

By increasing the breadth of the leading edge 31 of at least onesegregating step 11 (i.e., relative to the overall width of the spacewithin which the segregating passageway 101 is contained), capturedparticles will individually occupy a smaller percentage of the flow areaof the segregating passageway 101, reducing flow occlusion andincreasing the ability of the apparatus to maintain a near-constantthroughput. Constant throughput can reduce the need for complicated orexpensive fluid flow control equipment, since the pressure drop acrossthe apparatus should remain substantially constant so long as throughputremains substantially constant. A very broad step leading edge 31 cantherefore significantly reduce the tendency of the apparatus toexperience decreased throughput for samples having significant numbersof captured particles. Such apparatus can also capture a greater numberof size-segregated particles without exhibiting significantly decreasedthroughput.

The subject matter described herein is complementary to the subjectmatter disclosed in U.S. provisional patent applications no. 60/306,296,no. 61/125,168, no. 61/236,205, and no. 61/264,918 and in internationalpatent applications no. PCT/US2002/022689, no. PCT/US2009/002421, no.PCT/US2010/046350, and no. PCT/US2010/058172, each of which isincorporated herein by reference.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

For fluid flowing through a passageway in which a separation element 1as described herein is disposed, the “height” of the passageway is theminimum distance between the surfaces of the passageway between whichthe separation element 1 is interposed. For example, in each of FIGS. 1Dand 4C, a separation element 1 is interposed between a body 2 and acover 4. The minimum distance between the parallel faces of the body 2and cover 4 defines the height (h_(c)) of the passageway. Also visiblein these figures are the height (h0) of the passageways above thefocusing steps 10 of the separation elements 1 and the heights (h₁, h₂,and h₃) of the segregating passageways 101, 102, and 103 abovesegregating steps 11, 12, and 13, respectively. It is not critical thatthe ‘height’ dimension be oriented vertically relative to gravity duringoperation of the devices described herein.

A “focusing” step is merely a step which is disposed in (and preferablyextends most of the way or completely across) the channel on the inletside of a segregating step. A focusing step essentially directs fluidflow through the channel toward the portion of the narrow passagewaydefined by the segregating step, reducing potential areas of “deadvolume” in which little or no local fluid flow occurs. The channelshould have a greater height on the inlet side of the focusing step thanon its outlet side, such as with an inclined focusing step, or thefocusing step can have a more staircase-like conformation, includingmultiple steps. Devices described herein need not include a focusingstep, but inclusion of a separating step can be important in embodimentsin which minimization of dead volume (and retention therein of particlesintended to pass beyond the segregating step(s)) is desired.

The “width” of a passageway in which a separation element 1 as describedherein is disposed is the minimum distance, in the directionperpendicular to the direction of bulk fluid flow through the passageway(i.e., the overall general direction of such flow, ignoring localizedflow redirection induced by step geometries) and perpendicular to theheight of the passageway, between opposite faces of the passageway. Forexample, the width of a passageway is indicated as “W” in FIG. 7 foreach of four passageways containing steps of various geometries.

Further by way of example, the width dimension of the steppedpassageways 55 depicted in FIGS. 1D and 4C extend perpendicularly out ofthe figure. The “width” of a step is assessed in the same direction asthe width of a passageway in which the step is disposed; thus, the widthof a step that extends completely across the width a passageway is equalto the width of the passageway (even though the breadth of the leadingedge of the step may be significantly greater than the width of the stepowing, for example, to curvature or invagination of the leading edge).

The “breadth” of the leading edge 31 of a segregating step 11 is thelength of the leading edge 31, measured following the curvature of thestep. If the leading edge 31 is envisioned as an inflexible cord, thebreadth of the leading edge is the length of the cord when it is pulledtaut. Thus, the breadth of the leading edge of a curved or invaginatedstep can be significantly greater than the width of the step. This isillustrated in FIGS. 7A-7D, in which four leading edges 31 having alength 5W are configured in a variety of conformations, each leadingedge substantially exceeding the width (W) of the stepped passageway 55in which it is disposed.

The “broad face” of a step is the portion of a step that exists at atopographically altitude higher than a reference surface with respect towhich the step exists. The broad face of a step described herein willgenerally, but need not, be planar.

The “transitional face” of a step is the portion of the step thatbridges its broad face and the reference surface. The transitional facepreferably has a smooth or flat contour, and can be a surfaceperpendicular to both the reference surface and the broad face 41, asshown for transitional face 21 in FIG. 1. Transitional faces can also beinclined planar surfaces (see transitional face 20 in FIG. 6) or curved(see transitional face 20 in FIG. 5).

The “leading edge” 31 of a step is the portion of the step at which itsbroad face 41 meets its transitional face 21, for example as shown inFIG. 1.

The “flow area” of a passageway is a cross-section of the passagewaytaken in a plane perpendicular to the direction of bulk fluid flow inthe passageway.

Detailed Description

The disclosure relates to an apparatus for segregating particles on thebasis of their ability to flow through a segregating passageway 101having a minimum dimension (height) defined by the separation between asegregating step 11 of a separation element 1 and a surface of a void 50in which the separation element is disposed. The apparatus includes aseparation element 1 disposed in a void 50 formed by a body 2 and cover4. Within the void 50, the separation element 1 separates an inletregion 52 of the void from an outlet region 58 of the void. The inletand outlet regions are in fluid communication by way of a steppedpassageway 55 defined by the separation element 1 and one or both of thebody 2 and cover 4. One or more segregating steps 11 formed in theseparation element 1 define one or more segregation passageways 101.Fluid that flows between the inlet and outlet regions passes through thestepped passageway 55, including through at least a first segregationpassageway 101.

In operation, particles in an inlet region 52 of the void 50 pass intothe stepped passageway 55 and, if they are able, into the segregatingpassageway 101. Particles in the segregating passageway 101 can pass tothe outlet region 58 of the void 50. Cells that are not able to passinto or along the segregating passageway 101 do not reach the outletregion 58. In this way, particles able to reach the outlet region 58 aresegregated from particles that are not able to reach the outlet region58. The two populations of particles can be separately recovered fromthe apparatus. For example, particles at the outlet region 58 can berecovered in a stream of liquid withdrawn from the outlet region 58(e.g., by way of an outlet port or by way of a catheter inserted intothe outlet region 58). Particles unable to pass through the segregatingpassageway 101 to the outlet region 58 can be recovered by flushingthem, in the reverse direction, through the stepped passageway 55 andinto the inlet region 52. Such particles can be withdrawn from the inletregion 52. Alternatively, particles unable to pass through thesegregating passageway 101 to the outlet region 58 can be left in theapparatus or recovered by disassembling the apparatus.

The apparatus described herein can be used in a wide variety ofapplications. In addition to segregating particles from a mixedpopulation of particles, the device can be used in applications in whichone or more of the segregated particle populations are identified orfurther manipulated, for example. The construction and operation of theapparatus resist clogging by the particles being segregated, relative todevices previously used for particle separation.

By way of example, the apparatus can be used to isolate tumor cells froma mixed suspension of cells, such as to isolate circulating tumor cells(CTCs) present in the blood of a human or other vertebrate subject. Theapparatus can also be used to isolate fetal cells from the blood of awoman carrying (or who previously carried) a fetus. The apparatus canfurthermore be used to isolate from a mixed suspension of cellssubstantially any cell(s) that can be differentiated from others in thesuspension on the basis of their size, their compressibility, or acombination of these.

Parts and portions of the apparatus are now discussed separately ingreater detail.

The Body and Cover

The apparatus has a body 2 and a cover 4 defining a void 50therebetween. A portion of the void 50, defined in part by theseparation element 1, is a stepped passageway 55. The stepped passageway55 is also defined by a surface of the body 2, a surface of the cover 4,or by a combination of these, that is opposed to one or more steppedsurfaces (e.g., 31 and 32) of the separation element 1. In order tosimplify construction of the apparatus, most or all of the steppedpassageway-defining surfaces can be formed or machined into a separationelement 1 that is an integral part formed in a recess of the cover 4 orthe body 2, the recessed portion being surrounded by a flat surface, sothat the opposed surface of the body 2 or the cover 4 need only beanother flat surface in order to form the void 50 and enclose theseparation element 1 therein upon contact between the flat surfaces ofthe body 2 and cover 4.

The general format of the body 2 and cover 4 having an interposedseparation element 1 is discussed generally in documents that areincorporated herein by reference, and substantially any arrangementdescribed therein can be used for the apparatus described here.

Described herein are elements of the separation element 1 that are notdisclosed in those documents.

The body 2, the cover 4, or both can define an inlet port through whichfluid can be introduced into or withdrawn from the void 50. For example,the body 2 can define an inlet port that fluidly communicates with theinlet region 52. Fluid introduced into the inlet port can flow into theinlet region 52, displacing fluid already there (because the void issealed) into the stepped passageway, and thence sequentially into thefirst passageway 51, the second passageway 52, and the outlet region 58.Particles suspended in fluid in one of these regions and passageways canbe carried into a downstream region or passageway if the particle canflow through the present and intervening passageways and regions.Similarly, withdrawal of fluid from the outlet region 58 by way of anoutlet port formed in the body 2 can induce fluid flow from passagewaysin fluid communication with the outlet region 58 and from passagewaysand regions in fluid communication therewith.

Ports can be simple holes which extend through the cover or body, orthey can have fixtures (burrs, rings, hubs, or other fittings)associated with them for facilitating connection of a fluid flow deviceto the port. The body 2, cover 4, or both can define an inlet port inthe inlet region 52 of the void 50, an outlet port in the outlet region58 of the void 50, or both an inlet port and an outlet port. Fluid canbe introduced into the inlet region 52 through the inlet port. Fluid canbe withdrawn from the outlet region 58 through the outlet port.Continuous introduction of fluid into the inlet region 52 andsimultaneous withdrawal or emission of fluid from the outlet region 58can create a continuous flow of fluid through the apparatus. Similarly,continuous withdrawal of fluid from the outlet region 58 andsimultaneous influx or introduction of fluid into the inlet region 52can create continuous flow.

The Void

The body 2 and the cover 4 form a void 50 when they are assembled. Thevoid 50 has an inlet region 52, an outlet region 58, and a separationregion interposed between the inlet region 52 and the outlet region 58.A separation element 1 is disposed within the separation region and,together with the body 2, the cover 4, or both, defines a steppedpassageway 55. The stepped passageway 55 includes at least a firstsegregating passageway 101 that is defined by at least a firstsegregating step 11 in the separation element 1. The stepped passageway55 can include any number of additional segregating steps, each of whichcan define an additional segregating passageway in the void. Preferably,the only fluid path connecting the inlet and outlet regions 52 and 58 isthe stepped passageway 55, although that stepped passageway can beseparated into multiple stepped passageways, arranged in series, inparallel, or in some combination of these. Likewise, multiple devices asdescribed herein can be operated in series (e.g., to selectively captureparticles in selected size ranges) or in parallel (e.g., to enhance cellcapture capacity).

During operation of the device, at least the inlet region 52, the outletregion 58, and the stepped passageway of the void 50 are filled with afluid. Preferably, the entire void 50 is filled with fluid duringoperation. In one embodiment, the only fluid path that connects theinlet region 52 and the outlet region 58 is the stepped passageway.Particles present in the inlet region 52 can enter the steppedpassageway 55. The void (i.e., as defined by one or more of the body,cover, and separation element) can be formed so as to taper in thedirection of (or opposite) bulk fluid flow from the inlet region towardthe stepped passageway. Such void shapes can focus particles flow towardthe stepped passageway, maintain fluid linear flow velocity through theshaped region within a desired range (e.g., substantially constant),facilitate viewing of particles passing therethrough, or have otherbeneficial consequences.

Particles present in the stepped passageway 55 can enter and passthrough the first segregating passageway 101 unless they are excluded bythe height (i.e., the narrow dimension) of the first segregatingpassageway 101, or unless their movement through the first segregatingpassageway 101 is inhibited by particles which block that passageway(e.g., cells immobilized at or upstream from the leading edge 31 of thefirst segregating passageway 101. Particles which pass through the firstsegregating passageway 101 can enter the outlet region 58 and thence berecovered. Movement of particles within the apparatus can be induced byfluid flow through the apparatus, by intrinsic motility of the cells, ora combination of the two. Over time, particles unable to enter the firstpassageway 51 will be segregated in the inlet region 52; particles ableto traverse the first segregating passageway 101 will be segregated inor upstream from the stepped passageway 55; particles able to enter thefirst segregating passageway 101 but unable to freely move therethroughwill be segregated in the first segregating passageway 101; andparticles able to move through first segregating passageway 101 will besegregated in the outlet region 58 (or in fluid withdrawn or emittedfrom the outlet region 58).

Particles segregated in this manner can be recovered (using any of avariety of known methods, including some described herein) from theirrespective locations. By way of example, a catheter can be inserted intoa region or passageway (e.g., the inlet region 52 or first segregatingpassageway 101) of the apparatus, and particles present therein can bewithdrawn by inducing suction in lumen of the catheter. Further by wayof example, backflushing (i.e., fluid flow from the outlet region 58 inthe direction of the inlet region 52) can be used to collect particlespresent in one or more of the inlet region 52 or the first segregatingpassageway 101.

The Separation Element

The separation element 1 of the devices described herein can besubstantially the same as those described previously in U.S. Pat. No.7,993,908, in PCT publication WO 2011/066497, or elsewhere, but includesan additional feature. The separation element 1 of the devices describedherein include at least one segregating step 11 that has a leading edge31 with a breadth significantly greater than (e.g., 1.5×, 2×, 3×, 4×,5×, 10×, 20×, 50×, 100×, 500×, 1,000×, 10,000×, or 100,000× greaterthan) the overall width of a passageway within which the segregatingstep occurs. Put another way, the shape of the leading edge of at leastone step of the separation element 1 is such that the breadth of thatleading edge is substantially greater than the overall width of thestep. Put yet another way, the breadth of the leading edge of the step,assessed along its contour, is greater than the shortest linear distancebetween the two endpoints of the step edge (i.e., regardless of whetherthe step edge follows that shortest line). By way of example, theleading edge can be curved (see, e.g., FIG. 5), invaginated (see, e.g.,FIG. 6), angular (see, e.g., FIG. 4), serpentine (see, e.g., FIG. 3), orirregular (see, e.g., FIG. 5). The upper limit of the ratio of stepbreadth to passageway width is bounded substantially only by thetolerance of the manufacturing methods used to form the step and thesize of the particles that pass the step.

The stepped passageway 55 is the orifice through which particles move,fluid flows, or both, from the inlet region 52 to the outlet region 58during operation of the apparatus. The separation element 1 has astepped structure, which defines the stepped shape of at least one sideof the stepped passageway 55. The separation element 1 has at onesegregating step 11, and it can have multiple segregating steps (e.g.,11-13 in FIGS. 6-8). Fluid must flow through the segregating passageway101 defined in part by the corresponding segregating step 11 in order totraverse the stepped passageway 55 from the inlet region 52 to theoutlet region 58 when the apparatus is assembled.

During operation of the apparatus described herein, a mixture ofparticles having different sizes can be caused to flow through thestepped passageway 55, including at least one segregating passageway101. Passage of particles having a characteristic size in excess of thenarrow dimension (i.e., the height) of the segregating passageway 101 isimpeded at or near the leading edge 31 of the segregating step 11 thatbounds the segregating passageway, and such particles will tend toaccumulate at or near the leading edge 31 rather than passing throughthe segregating passageway 101. So long as the segregating passageway101 is not completely occluded by impeded particles across the entirebreadth of the segregating step 11, flow of fluid and particles aroundor past the impeded cells can continue. Development of the subjectmatter described herein arose, at least in part, as a result of attemptsto design apparatus less susceptible to fouling and clogging by impededparticles than prior art apparatuses. Preferably, the breadth ofseparating step 11 leading edges 31 are selected so that, for ananticipated mixture of particles, that the portion(s) of the segregatingpassageway 101 at which passage of particles are impeded has asufficient flow area that fluid flux through such portion(s) is notsignificantly (i.e., not more than 50%, 20%, 10%, 5%, 1%, 0.33%, or 0.1%or less) impeded when a desirable or foreseeable number of particles arelodged at the portion(s).

The separation element 1 can include a focusing step 10 (as illustratedin FIGS. 1-6), which serves to deflect fluid flow within the steppedpassageway 55 toward the first segregating passageway 101, to fill ‘deadspaces’ upstream of the first segregating step 11, to provide astructurally sound foundation for carrying segregating steps on theseparation element, or some combination of these. The separation neednot include a focusing step.

The steps of the separation element 1 can have any of a variety ofshapes. In one embodiment (e.g., in the apparatus depicted in FIG. 1),both the focusing step 10 and the first segregating step 11 have acommon ‘staircase-type’ step structure, i.e., two planar surfaces thatintersect at a right angle. That is, the transitional face 20 of thefocusing step 10 and the broad face 40 of the focusing step 10 meet at aright angle, as do the transitional face 21 of the first segregatingstep 11 and the broad face 41 thereof. Alternatively, the transitionaland broad faces of steps can meet at an angle between 90 and 180degrees, for example. The transitional and broad faces of the steps canalso meet at an angle between 0 and 90 degrees, forming an overhang. Forthe apparatus described herein, at least one segregating step 11 has acurved or, preferably, invaginated leading edge 31 and transitional face21, so that the breadth of the step is significantly greater than thewidth of the step.

Steps having transitional and broad faces that meet at an angle between90 and 180 degrees can occlude passage of particles having a variety ofsizes (i.e., those having sizes intermediate between the narrowdimension of the passageway defined by the broad face of the step andthe narrow dimension of the space upstream from the step. By haltingpassage of particles having slightly different sizes at differentpositions on the transitional face of the step, a step havingtransitional and broad faces that meet at an angle between 90 and 180degrees can prevent clogging of the passageway defined by the broad faceof the step to a greater degree than a step having transitional andbroad faces that meet at an angle of 90 degrees or less.

Clogging of fluid flow past a step by particles that occlude thepassageway defined by the broad face of the step can also be reduced oravoided by increasing the width of the step, as was recognized in theart. Because each particle occludes fluid flow only for the flow areaobscured by the particle, a wider step will necessarily be clogged by agreater number of occluding particles. However, increasing the width ofa step is not always practical, especially when significant widening isrequired to accommodate numerous particles or when miniaturization isdesired.

A significant aspect of the subject matter disclosed herein isrecognition by the present inventors that the capacity of a segregatingstep 11 to accommodate impeded particles can be significantly increasedwithout increasing the width of the step. Rather than (or in additionto) increasing the width of the segregating step 11, itsparticle-retention capacity can be increased by increasing the breadthof the leading edge 31 of the step (i.e., where particles impedanceoccurs), for example by decreasing the straightness of the step.

By way of example, in a fluid channel having a rectangularcross-section, a step that extends directly across (i.e., at rightangles to the sides) of the channel has a leading edge with a breadthsimply equal to the width of the channel (see, e.g., FIG. 1). If theshape of the step is a hemicircle, with the arc of the hemicircleextending such that the center of the hemicircle lies downstream fromthe upstream-most edge of the hemicircle, then the breadth of theleading edge of the step is equal to the perimeter of the hemicircle,which is the number pi multiplied by the width of the channel anddivided by two (i.e., roughly 1.57× the width of the channel).

Similarly, steps having leading edges shaped like an arc of a circle orellipse, like chevrons (i.e., like the letter V), like zig-zags, likeserpentine lines, or like irregular lines (See FIGS. 2-6) will all havebreadth values greater than the breadth of a step that simply extendsperpendicularly across a fluid channel having a rectangularcross-section. Steps having leading edges with such shapes can be usedin the apparatus described herein.

In one embodiment, the leading edge 31 of a segregating step 11 isshaped such that the breadth of the leading edge 31 is substantiallygreater (e.g., 1.5, 2, 3, 4, 5, 10, 20, 50, 100, or 1000 times greater)than the overall width of the step and/or the width of the segregationpassageway 101 defined by the step. This can be achieved, for example,by forming the step such that its leading edge has an undulating orhighly irregular edge shape, as illustrated in FIGS. 2 and 3, which arerepresentations of steps having undulating and irregular edges,respectively. In FIG. 2, the segregation step 11 is a flat slab havingfinger-shaped projections at its transition face 21. The breadth of theleading edge 31 of the step formed by the perimeter of the finger-shapedprojections is substantially greater than the width of the step, as canbe seen clearly in FIG. 2A. Likewise, the undulations and irregularitiesin the leading edge of the segregation step 11 illustrated in FIG. 3cause the breadth of the leading edge to be substantially greater thanthe overall width of the step, as can be seen clearly in FIG. 3A.

Multiple steps can have similarly- or differently-shaped leading edges.FIGS. 4-6, for example, illustrate separation elements 1 in which afocusing step 10 (which does not necessarily impede passage of anyparticles is shaped differently from each of segregating steps 11-13. Inthese illustration segregating steps 11-13 have the same or similarshapes, but they need not. Regardless of the shape of the leading edge31 of a segregating step 11, what is important to passage of cells orother particles through the segregating passageway 101 bounded by thesteps is the narrow dimension (height; e.g., h₁ in FIG. 1D) defined byeach segregating step 11. Particles unable to pass through the narrowdimension defined by a segregating step 11 will not traverse the step(unless it is able to deform and the pressure drop across the step issufficient to induce such deformation).

A series of segregating steps having progressively narrowing passagewaysdefined thereby, a segregating step having an inclined broad face (i.e.,so that the narrow passageway defined thereby narrows in the directionof bulk fluid flow therethrough), or a combination of these can be usedto capture deformable cells (i.e., cells which can deform to fit within,but not pass through, the passageway defined by a segregating step) andto segregate them from cells that are either sufficiently small orsufficiently deformable to pass the segregating step(s).

The breadth of each segregating step 11 can be selected based on theanticipated accumulation of particles on the step, in view of theparticle composition of sample anticipated to be processed using theapparatus and the narrow dimension of each corresponding segregatingpassageway 101. The breadth of a segregating step 11 can be selected tobe significantly (e.g., 10, 1,000, or 100,000 times) greater than thenarrow dimension of the corresponding segregating passageway 101. By wayof example, for segregation of fetal-like cells from maternal blood, abreadth approximately at least 1,000 (one thousand), and preferably10,000 (ten thousand), times the narrow dimension of the correspondingpassageway is considered desirable. Segregating steps 11 havingrelatively large breadth permit accumulation of particles within asegregating passageway 101 while limiting clogging of the segregatingpassageway 101.

Although the apparatus has been described herein with reference to asingle segregating step 11 (FIGS. 1-3 and 7) and with reference to threesegregating steps 11 (FIGS. 4-6), substantially any number ofsegregating steps 11 (e.g., two, four, ten, or one hundred steps) can beincluded in the apparatus, each segregating step 11 defining acorresponding segregating passageway 101 within the stepped passageway55 and having a characteristic narrow dimension.

Materials and Methods of Construction

The materials and methods used to make the devices described herein canbe substantially the same as those described previously in U.S. Pat. No.7,993,908, in PCT publication WO 2011/066497, or elsewhere, so long asthe leading edge 31 of at least one segregating step 11 of the apparatuscan be constructed as described herein—e.g., having a breadthsignificantly greater than its width, such as a leading edge 31 havingan undulating shape. That is, the methods must be able to make a devicehaving at least one segregating step 11 having a leading edge 31 breadthgreater than the overall width of the step (e.g., greater than the widthof a passageway within the device in which the step occurs).

Segregable Particles

The devices described herein can be used to segregate substantially thesame kinds of particles as those described previously in U.S. Pat. No.7,993,908, in PCT publication WO 2011/066497. Attributes of theparticles that affect their ability to traverse the segregationpassageway(s) 101 of the apparatus described herein include the size,shape, surface properties, and deformability of the particles.

In an important embodiment, the apparatus is used to segregate tumorcells (which tend to be significantly larger than correspondingnon-tumor cells of the same cell type) from non-tumor cells. It is knownthat tumor cells circulate in the bloodstream of many individual humans(as well as other vertebrate animals), even for tumors that areconsidered solid, unitary tumors, such as ovarian, prostate, and breastcancers. Detection and/or enumeration of circulating tumor cells (CTCs)can be an important indicator of the presence, nature (e.g., stage orgrade), malignancy, and response to treatment of a tumor. Furthermore,isolation of CTCs permits identification of the type of tumor that ispresent. These characteristics can be significantly important fordiagnosis, treatment, and prevention of metastasis of tumors.

In one embodiment, blood obtained from an individual (e.g., human)subject is processed using an apparatus described herein to segregateCTCs from the blood. Segregated CTCs can be recovered and analyzed byany known method to obtain important diagnostic, therapeutic, andpreventative information specific to the individual subject. BecauseCTCs are believed to be present even before development or establishmentof many advanced tumors, detection and characterization of CTCs canenable early, effective intervention to prevent tumor development andspread.

Substantially any diagnostic procedure amenable to use of isolated cellscan be performed using cells that are obtained from the device describedherein. Examples of such methods include assessing the affinity of anantibody preparation with such cells or an extract prepared from them,assessing nucleic acids contained within such cells, or assessing theability of the cells to grow in the presence of a selected medium or tointeract with other cells. Cells obtained using the devices describedherein can thus be used to assess gene expression, genetic changes,biomarker display, or other morphological or biochemical features of thecells (or changes to such features).

In another embodiment, the apparatus described herein is used tosegregate circulating endothelial cells (CECs) from a sample includingsuch cells, such a blood sample taken from a patient. CECs having anenlarged size (relative to normal CECs) can also be segregated byselecting appropriate narrow passageway dimensions in the apparatus. Byway of example, an apparatus can be used which has narrow passagewaydimensions selected to segregate enlarged CECs from normal CECs. Furtherby way of example, an apparatus can be used which has narrow passagewaydimensions selected to segregate all CEC (or only enlarged CECs) fromthe cells normally present in blood. CECs are known to be indicative ofthe presence or occurrence of trauma in an individual, and the presenceof enlarged CECs can be particularly indicative of certain conditions,such as acute or impending myocardial infarction (see, e.g., Damani etal., 2012, Sci. Transl. Med. 4:126ra33). CECs isolated using theapparatus described herein can also be recovered as described hereinand/or analyzed by conventional methods (e.g., by detection ofimmunological cell-surface markers) to identify their tissue of originand thereby further indicating the type and/or body location of thetrauma that induced their circulation. By way of example, isolation ofenlarged CECs of cardiac origin is indicative that the patient hasrecently undergone, is currently undergoing, or is imminently at riskfor occurrence of a myocardial infarction.

Fluid Displacement Devices

The apparatus described herein can be operated using substantially thesame types of fluid displacement devices as those described previouslyin U.S. Pat. No. 7,993,908, in PCT publication WO 2011/066497, or in theliterature pertaining to other microfluidic devices.

Using the Apparatus

Use and operation of the apparatus described herein are substantiallythe same as described previously in documents incorporated herein byreference. The apparatus described herein have the significant advantageof exhibiting less susceptibility to clogging, flow/throughputimpairment, and other undesirable phenomena attributable to capture ofcells on a segregating step 11 thereof.

EXAMPLES

The subject matter of this disclosure is now described with reference tothe following Examples. These Examples are provided for the purpose ofillustration only, and the subject matter is not limited to theseExamples, but rather encompasses all variations which are evident as aresult of the teaching provided herein.

In one embodiment, the stepped passageway has an overall width of 2.5centimeters and includes a second step 62 having an undulating leadingedge having a breadth of 8.0 centimeters. The narrow dimension of thesecond passageway 52 between the second step 62 and the opposed cover 4is 10 micrometers.

When a suspension of cells (e.g., 10 milliliters of human blood having aselected number of tumor cells included therein) is passed through thestepped passageway, followed by a rinsing solution that does not lysethe tumor cells, substantially all blood cells pass through theapparatus and most or all of the tumor cells are retained within it.

Table 1. Parts List

1 Separation Element

2 Body

4 Cover

10 Focusing Step

11 (First) Segregating Step

12 Second Segregating Step

32 Leading Edge of Second Segregating Step

13 Third Segregating Step

20 Transitional Face of Focusing Step

21 Transitional Face of (First) Segregating Step

22 Transitional Face of Second Segregating Step

23 Transitional Face of Third Segregating Step

30 Leading Edge of Focusing Step

31 Leading Edge of (First) Segregating Step

32 Leading Edge of Second Segregating Step

33 Leading Edge of Third Segregating Step

40 Broad Face of Focusing Step

41 Broad Face of (First) Segregating Step

42 Broad Face of Second Segregating Step

43 Broad Face of Third Segregating Step

50 Void defined by body and cover

52 Inlet Region of Void

53 Upstream Portion of channel

54 Channel connecting inlet and outlet regions of void

55 Separating Portion of channel

56 Downstream Portion of channel

58 Outlet Region of Void

60 Part of Separating Portion bounded by Focusing step

61 Part of Separating Portion bounded by (First) Segregating Step

62 Part of Separating Portion bounded by Second Segregating Step

63 Part of Separating Portion bounded by Third Segregating Step

101 (First) Segregating Passageway

102 Second Segregating Passageway

103 Third Segregating Passageway

Table 2. Abbreviations List

BFF Bulk Fluid Flow

hc Height of Channel

h0 Height of Channel in portion bounded by Focusing Step

h1 Height of Channel in portion bounded by (First) Segregating Step

h2 Height of Channel in portion bounded by Second Segregating Step

h3 Height of Channel in portion bounded by Third Segregating Step

W Overall Width of Channel in the Separating Portion

L Length of Separating Portion

B Breadth of Leading Edge of a Segregating Step

D ratio B/L

W Width of a Segregating Step

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

While the subject matter has been disclosed herein with reference tospecific embodiments, it is apparent that other embodiments andvariations of this subject matter can be devised by others skilled inthe art without departing from the true spirit and scope of the subjectmatter. The appended claims include all such embodiments and equivalentvariations.

1-32. (canceled)
 33. A method of segregating circulating tumor cells(CTCs) from blood cells in a fluid sample, wherein said method employs adevice comprising: a body and a cover that define a void therebetween,the void containing a separation element that segregates an inlet regionand an outlet region of the void, the separation element defining,together with a surface of the void, a channel that fluidly connects theinlet and outlet regions by way of a separating portion into which theseparation element projects, the channel having an overall width at theseparating portion and a height defined by the distance between theseparation element and the surface of the void, at least one of thebody, the cover or the separation element bearing a segregating stepdisposed within and having a leading edge extending substantiallycompletely across the separating portion of the channel, whereby thechannel is divided into an upstream portion on the inlet side of theleading edge and a substantially lamellar downstream portion on theoutlet side of the leading edge, the upstream portion of the channelbeing also lamellar in a region between the inlet region and theseparation element; the height of the upstream portion being sufficientto facilitate passage therethrough of both said CTCs and said bloodcells, the height of the downstream portion being sufficiently large tofacilitate passage therethrough of said blood cells and sufficientlysmall to inhibit passage therethrough of said CTCs, and the leading edgehaving a length greater than the overall width of the channel at theseparating portion; and said method comprises urging said fluid samplethrough said channel of said device from the inlet region whereby CTCswill be segregated from said blood cells due to their characteristicsand inability to traverse said segregating step along the separatingportion of the channel.
 34. The method as claimed in claim 33 whichfurther comprises recovering said segregated CTCs that are unable topass to the outlet region.
 35. The method as claimed in claim 34 whichfurther comprises backflushing fluid from the outlet region in thedirection of the inlet region to recover said segregated CTCs.
 36. Themethod as claimed in claim 33 wherein said CTCs are derived from a solidtumor.
 37. The method as claimed in claim 36 wherein said CTCs arederived from an ovarian, prostate or breast cancer tumor.
 38. The methodas claimed in claim 33 wherein said device has a size approximatelyequal to a microscope slide.
 39. The method as claimed in claim 33wherein said leading edge has an undulating or invaginated shape. 40.The method as claimed in claim 33 wherein the separation element of saiddevice includes a plurality of segregating steps disposed seriallywithin the separating portion, each segregating step: (a) having aleading edge that extends substantially across the separating portionand having a length substantially greater than the overall width of thechannel at the separating portion; and (b) dividing the channel into anupstream portion and a substantially lamellar downstream portionrelative to the leading edge of the segregating step, the height of thechannel at the downstream portion immediately following the segregatingstep being smaller than the height of the channel at the upstreamportion immediately preceding the segregating step.
 41. The method asclaimed in claim 39 wherein said leading edge has a serpentine shape.42. The method of claim 33 wherein said sample is a whole blood sample.43. A method of diagnosing occurrence of a tumor in a vertebratesubject, the method comprising the steps of: (i) segregating CTCs from ablood sample obtained from the subject using a method according to claim42 and (ii) determining the occurrence of thus segregated CTCs, whereinsaid occurrence of segregated CTCs is indicative of occurrence of atumor.
 44. The method as claimed in claim 43 wherein step (ii) includesexamining the portion of the device upstream of the leading edge of thesegregating step for segregated CTCs.
 45. The method as claimed in claim43 which further comprises performing a diagnostic test that assess acharacteristic of a tumor cell on one or more segregated CTCs.
 46. Amethod of assessing efficacy of a tumor treatment in a subject afflictedwith a tumor, the method comprising the steps of: (i) segregating CTCsfrom blood samples obtained from the subject before and after thetreatment using a method according to claim 42 and (ii) comparing theconcentration of CTCs in the samples from the CTCs thus segregated,whereby decrease of the concentration of CTCs after treatment isindicative of its effectiveness.